C H A P T E R F I V E 1 Fluorescence-Perturbation Techniques to Study Mobility and Molecular Dynamics of Proteins in Live Cells FRAP, Photoactivation, Photoconversion, and FLIP Aurélien Bancaud, Sébastien Huet, Gwénaël Rabut, and Jan Ellenberg Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany T HE TECHNIQUE OF FLUORESCENCE RECOVERY after photobleaching (FRAP) was introduced in the mid-1970s to study the diffusion of biomolecules in living cells (Peters et al. 1974; Edidin et al. 1976). For several years, it was used mainly by a small number of biophysicists who had devel- oped their own photobleaching systems. Since the mid-1990s, FRAP has gained increasing pop- ularity, because of the conjunction of two factors: First, photobleaching techniques are easily implemented on confocal laser-scanning microscopes (CLSMs) (McNally and Smith 2001), and so FRAP has become available to anyone who has access to such equipment. Second, the advent of green fluorescent protein (GFP) has allowed easy fluorescent tagging of proteins and their observation in living cells (see Chaps. 1 and 2). Thanks both to the versatility of modern CLSMs, which allow control of laser intensity at any point of the image, and to the development of new fluorescent probes, other photoperturbation techniques have emerged during the last few years. In essence, all these techniques aim to alter the steady-state fluorescence distribution in a specimen by photoperturbing fluorescence in selected regions. Such approaches include photobleaching, photoactivation (i.e., when nonfluorescent probes become fluorescent after illumination at a given wavelength), and photoconversion (i.e., when irradiation of fluorophores at a given wavelength induces a translation of their fluorescence spectrum toward longer wavelengths) (Fig. 1). After the photoperturbation event, one observes and then analyzes how the fluorescence distribution relaxes toward the steady state. Because the photochemical perturbation of suitable fluorophores is essentially irreversible, changes of fluores- cence intensity in the perturbed and unperturbed regions are due to the exchange of tagged mol- ecules between those regions. For the sake of simplicity and to give a generic name for these dif- ferent techniques, the FRAP acronym will hereafter refer to fluorescence redistribution after photoperturbation instead of its original meaning. In a typical FRAP experiment (see Fig. 1) a small region of the fluorescent specimen is photo- perturbed once. The images are analyzed to display the subsequent variations of the fluorescence signal in the photoperturbed region. FRAP experiments are easy to set up and thus are very popu- lar. They give qualitative information about the behavior of molecules in the photoperturbed region (i.e., whether they are mobile or immobile). Moreover, a comparison of the characteristic